scholarly journals Selection of a suitable viral DNA extraction method for Sheeppox virus in cell culture

2021 ◽  
Vol 26 (6) ◽  
pp. 3095-3101
Author(s):  
PIYALI MONDAL ◽  
C L PATEL ◽  
RACHNA SAGAR ◽  
INSHA ZAFIR ◽  
JOYSHIKH SONOWAL ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Isoamyl Alcohol ◽  
Good Method ◽  
Proteinase K ◽  
Acid Extraction ◽  
Limited Information ◽  
Sheeppox Virus

A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p<0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

Effects of diverse materials-based methods on DNA extraction for Clostridium difficle from stool samples

2019 ◽  
Vol 9 (5) ◽  
pp. 509-516 ◽  
Author(s):  
Ziqi Xiao ◽  
Gaojian Yang ◽  
Deng Yan ◽  
Song Li ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Magnetic Beads ◽  
Diagnostic Technique ◽  
Proteinase K ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Spin Column ◽  
Stool Samples

Nosocomial infections, including Clostridium difficile infection (CDI), and their fatality rates have increased in the past few decades. Despite emerging molecular diagnostic technologies with rapid, accurate outcomes, nucleic acid extraction from stool samples remains the first limiting step before downstream applications. Commercial nucleic acid extraction kits greatly decrease labor and time requirements, and also provide nucleic acid preparations with higher quality and purity for enzyme digestion analysis or genotyping. The magnetic bead based technique is a novel method compared with the conventional spin-column method, and currently has widespread use in nucleic acid extraction. We evaluated five DNA extraction kits with magnetic beads using materials with various properties (particle size, concentration of magnetic beads, grinding beads) and reagents (proteinase K, lysozyme, isopropanol, and absolute ethanol) to determine the cost, hands-on time, number of essential operations, and quality and purity of the DNA preparations, compared with those obtained using the QIAamp Fast DNA Stool Mini Kit. The six DNA extraction kits yielded A260/280 ratios ranging from 0.85 to 1.9 (average 1.57), and concentrations from 3.70 to 108.09 ng/μL (average 34.64 ng/μL). All the DNA samples had acceptable downstream application effects, except for those obtained using the TIANGEN Magnetic Soil and Stool DNA Kit. However, gel electrophoresis analysis of the DNA samples resulted in a light strip on the gel, indicating that the proteinaceous contaminant may not have been removed completely. A rapid and accurate molecular diagnostic technique could allow for more suitable treatment and prognosis outcomes for inpatients, depending, in large part, on the quality and purity of DNA preparations, which are frequently neglected. Our study focused on the quality of commercial kits with a primary focus on the treatment of stool samples and molecular diagnostic applications.

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

scholarly journals Methodological Approaches to DNA Authentication of Foods, Wines and Raw Materials for Their Production

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 595
Author(s):  
Aram G. Galstyan ◽  
Vladislav K. Semipyatniy ◽  
Irina Yu. Mikhailova ◽  
Khamid Kh. Gilmanov ◽  
Alana V. Bigaeva ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Materials ◽  
Direct Sequencing ◽  
Genetic Identification ◽  
Proteinase K ◽  
Vitis Vinifera L ◽  
Acid Extraction ◽  
Plant Component ◽  
Sensitivity Specificity

DNA authentication of wines is a process of verifying their authenticity by genetic identification of the main plant component. The sample preparation of experimental and commercial wines was carried out by precipitation of wine debris by centrifugation with preliminary exposure with precipitators and co-precipitators, including developed macro- and micro- volume methods applicable to white or red wines, using polyvinylpyrrolidone as a co-precipitator. Addition of 2-mercaptoethanol and proteinase K to the lysing solution made it possible to adapt the technology for DNA extraction from the precipitated wine debris. The additionally tested technique of DNA extraction from wine debris by dimethyl sulfoxide (DMSO) lysis had fewer stages and, consequently, a lower risk of contamination. The results of further testing of one of the designed primer pairs (UFGT-F1 and UFGT-R1) in conjunction with the tested methods of wine material sample preparation and nucleic acid extraction, showed the advantage in the given set of oligonucleotides over previously used ones in terms of sensitivity, specificity and reproducibility. The developing strategy for genetic identification of grape varieties and DNA authentication of wines produced from them based on direct sequencing of polymerase chain reaction (PCR) products is implemented by interpreting the detected polymorphic positions of variable Vitis vinifera L. UFGT gene locus with distribution and split into 13 UFGT gene-associated groups.

scholarly journals Extracting DNA of nematodes communities from Argentine Pampas agricultural soils

2015 ◽  
Vol 87 (2) ◽  
pp. 691-697
Author(s):  
Eduardo A. Mondino ◽  
Fernanda Covacevich ◽  
Guillermo A. Studdert ◽  
João P. Pimentel ◽  
Ricardo L.L. Berbara
Keyword(s):  
Dna Extraction ◽  
Cetyltrimethylammonium Bromide ◽  
Agricultural Soils ◽  
Sodium Dodecyl ◽  
Isoamyl Alcohol ◽  
Proteinase K ◽  
Chelex 100 ◽  
Polymerase Chain ◽  
Argentine Pampas

We examined four strategies (Tris/EDTA, sodium dodecyl sulfate, Chelex 100 resin and cetyltrimethylammonium bromide -CTAB-) for extracting nucleic acid (DNA) from communities of nematodes. Nematodes were isolated from an agricultural area under different management of long-term crop rotation experiment from Argentina during three seasons. After DNA extraction, Polymerase Chain Reaction-amplifications were performed and considered as indicators of successful DNA extraction. The CTAB combined with proteinase K and phenol-chloroform-isoamyl alcohol was the unique successful method because positive amplifications were obtained by using both eukaryotic and nematode specific primers. This work could contribute to biodiversity studies of nematodes on agroecosystems.

scholarly journals Comparison of Six Commercial DNA Extraction Kits for Recovery of Cytomegalovirus DNA from Spiked Human Specimens

2000 ◽  
Vol 38 (10) ◽  
pp. 3860-3863 ◽  
Author(s):  
Gary A. Fahle ◽  
Steven H. Fischer
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Cerebral Spinal Fluid ◽  
Processing Time ◽  
Dna Isolation ◽  
Dna Purification ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Inhibitors ◽  
Positive Results

We evaluated six commercially available DNA extraction kits for their ability to recover DNA from various dilutions of cytomegalovirus (CMV) added to four different specimens: bronchoalveolar lavage, cerebral spinal fluid, plasma, and whole blood. The kits evaluated included the Puregene DNA isolation kit (PG), Generation Capture Column kit, MasterPure DNA purification kit, IsoQuick nucleic acid extraction kit, QIAamp blood kit, and NucliSens isolation kit (NS). All six kits evaluated effectively removed PCR inhibitors from each of the four specimen types and produced consistently positive results down to a spiked concentration of 200 PFU of whole CMV per ml. However, the NS and PG resulted in the most consistently positive results at the lowest concentrations of spiked CMV (4 and 0.4 PFU/ml) and, in this evaluation, offered the most sensitive methods for extracting CMV DNA from the four different spiked specimens. Processing time and cost were also evaluated.

scholarly journals SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

2020 ◽  
Author(s):  
Chantal B.F. Vogels ◽  
Anne E. Watkins ◽  
Christina A. Harden ◽  
Doug Brackney ◽  
Jared Shafer ◽  
...  
Keyword(s):  
Supply Chain ◽  
Nucleic Acid ◽  
Large Scale ◽  
Limit Of Detection ◽  
Proteinase K ◽  
Acid Extraction ◽  
Sample Collection ◽  
Reverse Transcription Pcr ◽  
August 15Th ◽  
Frequent Sampling

Current bottlenecks for improving accessibility and scalability of SARS-CoV-2 testing include diagnostic assay costs, complexity, and supply chain shortages. To resolve these issues, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration on August 15th, 2020. The critical component of our approach is to use saliva instead of respiratory swabs, which enables non-invasive frequent sampling and reduces the need for trained healthcare professionals during collection. Furthermore, we simplified our diagnostic test by (1) not requiring nucleic acid preservatives at sample collection, (2) replacing nucleic acid extraction with a simple proteinase K and heat treatment step, and (3) testing specimens with a dualplex quantitative reverse transcription PCR (RT-qPCR) assay. We validated SalivaDirect with reagents and instruments from multiple vendors to minimize the risk for supply chain issues. Regardless of our tested combination of reagents and instruments from different vendors, we found that SalivaDirect is highly sensitive with a limit of detection of 6-12 SARS-CoV-2 copies/μL. When comparing SalivaDirect to paired nasopharyngeal swabs using the authorized ThermoFisher Scientific TaqPath COVID-19 combo kit, we found high agreement in testing outcomes (>94%). In partnership with the National Basketball Association (NBA) and Players Association, we conducted a large-scale (n = 3,779) SalivaDirect usability study and comparison to standard nasal/oral tests for asymptomatic and presymptomatic SARS-CoV-2 detection. From this cohort of healthy NBA players, staff, and contractors, we found that 99.7% of samples were valid using our saliva collection techniques and a 89.5% positive and >99.9% negative test agreement to swabs, demonstrating that saliva is a valid and noninvasive alternative to swabs for large-scale SARS-CoV-2 testing. SalivaDirect is a flexible and inexpensive ($1.21-$4.39/sample in reagent costs) option to help improve SARS-CoV-2 testing capacity. Register to become a designated laboratory to use SalivaDirect under our FDA EUA on our website: publichealth.yale.edu/salivadirect/.

scholarly journals Optimization of genomic DNA extraction protocol for black wattle

Agrarian ◽  
2020 ◽  
Vol 13 (49) ◽  
pp. 323-329
Author(s):  
Yasmin Imparato Maximo ◽  
Angela Cristina Ikeda ◽  
Paulo César Flôres Júnior ◽  
Giovana Bomfim De Alcantara ◽  
Antonio Higa
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Proteinase K ◽  
Genomic Dna Extraction ◽  
Extraction Protocol ◽  
Black Wattle

Considerando-se que a atual tendência do melhoramento florestal é a integração das técnicas clássicas com as de análise genética molecular, faz-se necessária a obtenção de protocolos de extração de DNA genômico ajustados a cada espécie estudada. O objetivo do trabalho foi determinar o efeito de diferentes adaptações no protocolo de extração de DNA genômico CTAB para acácia-negra. Foram testados diferentes componentes na fase de extração orgânica: clorofórmio, fenol e proteinase K, além da aplicação de RNase após a fase de precipitação e limpeza do DNA. Também, foi investigada a eficiência destes tratamentos em amostras de folíolos frescas ou armazenadas em baixa temperatura durante sete dias. Foi verificada a presença de DNA de todas as amostras submetidas à extração pelo protocolo de CTAB com os diferentes tratamentos. O tempo de armazenamento das amostras não influenciou na integridade do DNA, entretanto, foi possível observar que a adição de RNase melhorou a qualidade do DNA extraído. Deste modo, sugere-se a utilização do protocolo CTAB com uso de clorofórmio e RNase, com amostras frescas ou armazenadas em baixas temperaturas.

Research note: Rapid isolation of total RNA and genomic DNA from Hakea actities

2002 ◽  
Vol 29 (8) ◽  
pp. 1015 ◽  
Author(s):  
Michael G. Mason ◽  
Susanne Schmidt
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Genomic Dna ◽  
Rna Extraction ◽  
Nutrient Concentrations ◽  
Cluster Roots ◽  
Acid Extraction ◽  
Native Plant ◽  
Nucleic Acid Extraction ◽  
Total Rna

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

scholarly journals Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains

1970 ◽  
Vol 4 (1) ◽  
pp. 53-57 ◽  
Author(s):  
Mohammad Shahriar ◽  
Md Rashidul Haque ◽  
Shaila Kabir ◽  
Irin Dewan ◽  
Mohiuddin Ahmed Bhuyian
Keyword(s):  
Bacillus Subtilis ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Procedure ◽  
Proteinase K ◽  
Clinical Samples ◽  
Gram Positive ◽  
Genomic Dna Extraction ◽  
Dna Concentration ◽  
The Mean

Direct extraction of DNA from natural environment and clinical samples has become a useful alternative for the phylogenetic identification and in situ detection of individual microbial cells without cultivation. In this study, three different Gram positive microorganisms (B. cereus, B. subtilis, and S. aureus) were chosen for genomic DNA extraction. High salt SDS (Sodium Dodesyl Sulfate) based extraction method was followed to extract genomic DNA with addition of three different lysis protocols to observe the effect of proteinase-K on total genomic DNA yield, lysis steps were carried with SDS, SDS with 3 μl proteinase-K and SDS with 6μl proteinase-K. High molecular weight intact DNA bands were observed only for Bacillus subtilis when the extraction procedure was carried out in presence of SDS, SDS with proteinase-K (3μl) and SDS with increased amount of proteinase-K (6μl). In presence of SDS and increased amount of proteinase-K (6μl) the mean value of DNA concentration for Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus were found to be 1.53±0.15, 1.36±0.10 and 1.65±0.10 μg/μl respectively. However, in absence of proteinase-K, the mean values of DNA concentration were found to be decreased (1.28±0.10, 1.34±0.15, 1.23±0.10 μg/μl for B. cereus, B. subtilis, and S. aureus respectively) for all these stains. Although in case of B. subtilis the overall effect of proteinase-K was not found to be significant in terms of DNA concentration and DNA band intensity, however, for B. cereus, and S. aureus sharp decrease in total extracted DNA concentration was observed suggesting the increased lysis effect of proteinase-K on the thick peptidoglycan layer of Gram-positive cell wall such as B. cereus, and S. aureus.   Key words: Extraction; Genomic DNA; Lysis buffer; Gram positive organism. DOI: http://dx.doi.org/10.3329/sjps.v4i1.8867 SJPS 2011; 4(1): 53-57

Self-powered switch-controlled nucleic acid extraction system

Lab on a Chip ◽  
2016 ◽  
Vol 16 (1) ◽  
pp. 132-141 ◽  
Author(s):  
Kyungsup Han ◽  
Yong-Jin Yoon ◽  
Yong Shin ◽  
Mi Kyoung Park
Keyword(s):  
Nucleic Acid ◽  
Dna Extraction ◽  
Extraction System ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
System P ◽  
Self Powered ◽  
User Friendly

We have developed a Self-powered Switch-controlled Nucleic acid Extraction System (SSNES) to overcome the limitation of LOC technology in POC applications. The SSNES have a potential to be widely used as powerless, fully-disposable and user-friendly system for DNA extraction.

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